导致采后龙眼果实腐烂的拟茎点霉产胞壁降解酶、角质酶及致病机理研究

Longan (Dimocarpus longan Lour.) is a famous subtropical fruit species in South China, but the fruit deteriorates rapidly after harvest due to rot development caused by Phomopsis longanae Chi. However, little information is available on the pathogenesis of Phomopsis longanae Chi. causing fruit decay in harvested longans..The objective of this study is to clarify the major degrading-enzymes produced by P. longanae Chi. and its role in pathogenicity to harvested longans during fruit-rotting development, the kinds and activities of degrading-enzymes such as cell wall-degrading enzymes and cutinase produced by P. longanae Chi. in vitro, and the affecting factors of culture conditions on the kinds and activities of degrading-enzymes produced by P. longanae Chi. in vitro, and the degrading-enzymes produced by P. longanae Chi. in vitro to the damage of cell membrane, tissue maceration and the destruction of the structures of cell and tissue of harvested longan fruits are investigated..The another objective of this study is to reveal the roles of degrading-enzymes produced by P. longanae Chi. causing fruit decay in harvested longans during storage, the activities of degrading-enzymes such as cell wall-degrading enzymes and cutinase in different parts of the lesion of fruits （healthy area, boundary between diseased and healthy area, diseased area）caused by P. longanae Chi., and the changes of activities of degrading-enzymes such as cell wall-degrading enzymes and cutinase in P. longanae Chi.-inoculated longans during fruit rot development in relation to the destruction of cell and tissue structures such as breakdown of cuticle and cell wall are also conducted..The results are expected to obtain a new theoretical basis for elucidating the possible mechanism of fruit rot development caused by P. longanae Chi., and further to provide a scientific basis for controlling the rot development of harvested longan fruit.

龙眼拟茎点霉（Phomopsis longanae Chi.）是引起采后龙眼果实腐烂最主要的病原菌。目前对拟茎点霉引起龙眼果实腐烂的机制所知甚少。本项目拟从离体培养（活体外）条件下龙眼拟茎点霉产降解酶（胞壁降解酶、角质酶）种类和活性，培养条件对该病原菌产降解酶种类和活性的影响，及该菌产生的降解酶对龙眼果实细胞膜损伤、组织浸解、细胞组织结构破坏的影响等方面阐明龙眼拟茎点霉产降解酶的主要种类及其在该病原菌致病中的作用；同时研究拟茎点霉接种后的龙眼果实（活体内）在不同感病部位（健处、病健交界处、病斑处）其降解酶活性的差异，及经拟茎点霉接种的龙眼果实在腐烂过程中降解酶活性变化与果实角质层、细胞壁降解等细胞组织结构破坏的关系，以揭示拟茎点霉产降解酶在该菌所致龙眼果实腐烂进程中的作用。研究成果可望在阐明拟茎点霉侵染所致龙眼果实腐烂发生的可能机制获得新的理论依据，进而为控制采后龙眼果实腐烂提供科学依据。

本项目通过体内和体外试验互相验证的方法，明确了龙眼拟茎点霉产降解酶在拟茎点霉所致龙眼果实腐烂发生进程中的作用。结果如下：.1、研究离体培养（活体外）条件下P. longanae产降解酶的种类、活性及条件优化。P. longanae在改良的Marcus液体培养基中可以产Cx、β-葡萄糖苷酶、PG和PMG四种主要的细胞壁降解酶。如P. longanae产Cx最佳条件为：培养时间为2 d，培养基初始pH为6.5，碳源为龙眼壳粉1.5%，氮源为硝酸钾2%。.2、研究离体培养（活体外）P. longanae产果胶酶、纤维素酶等单一降解酶或混合酶对龙眼果实细胞膜损伤、组织浸解、组织结构破坏的影响。龙眼果实经果胶酶、纤维素酶和混合酶处理后，龙眼果皮细胞膜透性增加，损伤影响以混合酶处理的最严重，损伤率高达48.37%，而纤维素酶和果胶酶损伤率分别为40.43%和34.78%。进一步观察发现，单一酶或混合酶处理的龙眼果皮组织均会出现不同程度的破坏作用，呈褐色水渍状，其中以混合酶处理的破坏程度最大，果胶酶次之，纤维素酶最小。.3、研究经P. longanae接种的龙眼果实（活体内）不同感病部位（健康处、病健交界处、病斑处）降解酶活性的差异性。P. longanae接种龙眼果实后在寄主体内也能产生Cx、-葡萄糖苷酶、PG和PMG四种细胞壁降解酶，且不同部位的酶活性差异很大。以病健交接处酶活性最高，其次是病斑处，健康处的酶活性最低。.4、研究P. longanae接种的龙眼果实在腐烂过程中降解酶活性变化与果皮细胞壁组分降解的关系。P. longanae侵染加速龙眼果皮细胞壁结构的破坏，导致果皮软化、组织疏松，主要表现为P. longanae接种的龙眼果实果皮PMG、PG、Cx和β-葡萄糖苷酶活性以及水溶性果胶含量增加，而果皮原果胶、纤维素和半纤维素含量下降。因此认为，P. longanae侵染促进龙眼果皮败坏与提高龙眼果皮细胞壁降解酶活性而加速果皮细胞壁物质的降解有关。.5、研究P. longanae侵染对龙眼果实采后果皮褐变、果实病害的影响及其与能荷水平的关系。与对照果实相比，P. longanae侵染提高了龙眼果皮褐变指数和果实病害指数。P. longanae侵染促进龙眼果皮ATP、ADP含量和能荷水平下降，AMP含量上升。因此认为，龙眼果实采后果皮褐变、病害发生与能量亏缺有关。